The reasearch being carried out involves investigations of the in vivo and in vitro metabolism of cyclophosphamide (CP) in an effort to define the role of metabolism in chemotherapy and toxicity of CP and understand the mechanism by which CP produces specific depression of hepatic drug metabolism. In vitro studies on the binding of 3H-chloroethyl CP and 14(C-4)-CP suggested that acrolein binds to microsomal proteins via interaction with sulfhydryl groups in proteins. This interaction causes the denaturation of cytochrome P-450 which can be blocked by chemicals with free sulfhydryl groups, e.g. glutathione, cysteine, N-acetyl cysteine. Patterns of in vivo macromolecular binding of 14(C-4)-CP in glutathione depleted or cysteine-treated mice demonstrated that the binding of the 14C-label to proteins increases in glutathione-depleted animals. Futhermore, both CP and acrolein caused dose-, and time-dependent depletion of glutathione in mice, whereas phosphoramide mustard was relatively ineffective. CP was also more toxic to glutathione-depleted rats and mice and the toxicity of CP was significantly decreased by pretreatment with cysteine. Studies on the interaction between CP metabolites and nucleic acids and on the nature of these interactions are in progress.